Amphiphilic block copolymers, having a large solubility difference between hydrophilic and hydrophobic blocks, are known to assemble in aqueous solutions into polymer micelles of nanoscale size (see, e.g., Moffitt, M., et al. Acc. Chem. Res. 29: 95-102 (1996)). Such micelles have a fairly narrow size distribution and are characterized by their unique core-shell architecture, where hydrophobic blocks are segregated from the aqueous exterior to form an inner core surrounded by a shell of hydrophilic polymer chains. A micelle is thermodynamically stable relative to disassembly into single chains as long as the concentration of the block copolymer exceeds the critical micelle concentration (CMC).
Aggregation number, size, and shell architecture parameters of polymer micelles are essential characteristics for determining whether micelles will be useful in pharmaceutical applications. In general, the hydrophobic micelle core serves as a microenvironment for the incorporation of various therapeutic or diagnostic reagents while the hydrophilic shell or exterior stabilizes the micelles in aqueous dispersion. Poly(ethylene oxide) (PEO) is frequently used as the hydrophilic block of micelle-forming copolymers, since this polymer is known to be highly hydrated, soluble, non-toxic, non-immunogenic, and is able to serve as an efficient steric protector for various microparticulates such as liposomes, nanoparticles, or nanocapsules in biological media (see, e.g., Kwon, G. S, and Kataoka, K., Adv. Drug Deliv. Rev. 16: 295-309 (1995); and Tobio, M., et al. Pharm. Res. 15: 270-275 (1998)). In particular, PEO chains prevent particle opsonization (phagocytosis or degradation), rendering the particles “unrecognizable” by the reticuloendothelial system. In addition, polymer micelles that range in size between about 10 nm and about 150 nm evade renal excretion and non-specific capture by the reticuloendothelial system, and demonstrate prolonged circulation times in blood (see, e.g., Stolnik, S., et al., Adv. Drug Deliv. Rev. 16: 195-214 (1995); and Kataoka, K., et al. J. Controlled Rel. 24: 119-132 (1993)).
The nanoscale size of some polymer micelles facilitates “passive targeting,” which results in significant accumulation of the micelles in tumor tissue, known as the EPR effect (again see Kwon, G. S, and Kataoka, K., Adv. Drug Deliv. Rev. 16: 295-309 (1995); and Kataoka, K., et al. J. Controlled Rel. 24: 119-132 (1993)). This EPR effect has been attributed to leaky tumor vessels that allow in particle extravasation in tumor sites, where there is no such extravasation in normal tissues. Thus, EPR and lack of effective tumor lymphatic drainage prevent clearance of polymer micelles and promote accumulation of micelles in tumors (see Maeda, H., Ad. Enzyme Regul. 41: 189-207 (2001); and Duncan, R., Pharm. Sci. Techn. Today 2: 441-449 (1999)).
The aforementioned beneficial properties of polymer micelles can be successfully exploited for drug delivery, particularly in cancer as a tumor-specific delivery system. For example, in vivo studies have shown that the life span of animals and inhibition of tumor growth were increased considerably in mice treated with a drug incorporated in block copolymer micelle (see, e.g., Yokoyama, M., et al., J. Controlled Release 50: 79-92 (1998); and Kabanov, et al., W098/56334; and Kabanov et al., W098/56348)). Furthermore, recent studies have demonstrated that incorporation of antracyclines and other cytotoxic drugs in pluronic block copolymer micelles considerably can reduce drug resistance of various tumor cells (see Kabanov, A. V. and Lakhov, V., Cr. Rev. Ther. Drug Targ. 19: 1-73; and Kakizawa, Y., et al. J. Am. Chem. Soc. 121: 11247-11248 (1999)). Indeed, more than a 1000-fold increase in sensitivity of resistant tumors was observed for doxorubicin-loaded pluronic micelles (Alakhov, V., et al. Colloids Surf., B: Biointerfaces 16: 113-134 (1999)). A Phase II clinical trial of doxorubicin formulated with pluronic block polymer is currently being conducted (see Ranson, M., et al., The 5th International Symposium on Polymer Therapeutics: From Laboratory to Clinical Practice, The Welsh School of Pharmacy, Cardiff University, Cardiff, UK, p. 15 (2002)). Together these studies suggest that incorporation of anticancer drugs in polymer micelles can increase the efficacy of cancer chemotherapy.
Factors that influence the performance of polymer micelles for drug delivery are loading capacity, release kinetics, circulation time, biodistribution, size, and stability (see, e.g., Allen, C., et al. Colloids Surf., B: Biointerfaces 16: 3-27 (1999)). Studies have shown that the in vivo anti-tumor activity of a drug is positively correlated with its in vitro stability. Therefore, a micelle structure with high stability is desired. One clear reason is that a delivery system is subject to severe dilution upon intravenous injection into an animal or human subject. In the bloodstream, under dilution, multimolecular micelles formed by block copolymers disintegrate or dissolve causing changes in their structure and size. Such instability of micelles is one concern for their application in vivo.
Formation of cross-links between the polymer chains of the core domain introduces covalent stabilization that reinforces micellar architecture and results in high stability against dilution and shear forces. The cross-linking results, in essence, in single molecules of nanoscale size. Therefore, in contrast to prior art supramolecular micelles, the nanoscale micelles of the present invention do not disintegrate under environmental variations such as dilution, changes in ionic strength or solvent system or pH.
There are several reports on the stabilization of the polymer micelles by cross-linking either within the core domain (see Iijima, M., et al., Macromolecules 32: 1140-1146 (1999); Kim, J.-H., et al., Polym. Adv. Technol. 10: 647-654 (1999); Won, Y.-Y., et al., Science 283: 960-963 (1999); Guo, A., et al., Macromolecules 29: 2487-2493 (1996); Wooley, et al., U.S. Pat. No. 6,383,500; Kabanov, et al., U.S. Pat. No. 6,333,051; and Rapoport, N., Colloids Surf, B: Biointerfaces 16: 93-111 (1999)) or throughout the shell layer (Thurmond, K. B. II, et al., Colloids Surf., B: Biointerfaces 16: 45-54 (1999); Wooley, et al., U.S. Pat. No. 6,383,500; Zhang, O., et al., J. Am. Chem. Soc. 122: 3642-3651 (2000); and Bütün, V., et al., J. Am. Chem. Soc. 121: 4288-4289 (1999)). In these cases, the cross-linked micelles maintained small size and core-shell morphology while their dissociation was suppressed. For example, stable nanospheres were prepared from poly(ethylene glycol)-b-polylactide micelles by using a polymerizable group at the core segment (again see Iijima, M., et al., Macromolecules 32: 1140-1146 (1999)); which, in addition to stabilization against temperature change and time passage, obtained core polymerized micelles that exhibited excellent solubilization of rather large molecules such as taxol.
The incorporation of therapeutics or diagnostics (pharmaceutically active agents) into polymer micelles may be achieved through chemical and physical routes. Chemical routes generally involve covalent coupling of the drug to the hydrophobic block of the copolymer, leading to micelle-forming, polymer-drug conjugates (see, e.g., Kataoka, K., et al. Adv. Drug Del. Rev. 47: 113-131 (2001); Kataoka, K., et al., J. Controlled Rel. 24:119-132 (1993); Bader, H., et al., Chem. 123/124: 457-483; and Yokoama, M., et al., Cancer Res. 50: 1693-1700 (1990)). However, the physical encapsulation of drugs within the polymer micelle (also known as micellar nanocontainers) is generally a more attractive procedure and currently is being used for a wide variety of micellar systems because it is simpler and facilitates drug release (see Kabanov, A. V., et al., FEBS Lett. 258: 343-345 (1989); Hagan, S. A., et al., Langmuir 12: 2153-2163 (1996); and Kwon, G. S., et al. Colloids Surf, B: Biointerfaces 2: 429-434 (1994)).
The principles of the self-assembly of polymer micelles have been significantly advanced by using block and graft (linear polymers to which side chains have been “grafted”) copolymers containing ionic and nonionic blocks. Upon interaction with oppositely charged polyions or surfactants, a special class of dispersed systems are formed, known as polymer complex micelles or block ionomer complexes, which combine the properties of polyelectrolyte complexes and block copolymer micelles (see, e.g., Harada, A. and Kataoka, K., Macromolecules 28: 5294-5299 (1995); Kabanov, A. V., et al., Macromolecules 29: 6797-6802 (1996); Bronich, T. K., et al., Macromolecules 30: 3519-3525 (1997); Bronich, T. K., et al., Colloids Surf., B: Biointerfaces 16: 243-251 (1999); and Harada, A., et al., Macromolecules 31: 288-294 (1998)). Micelles formed from block or graft copolymers are of interest because they allow the encapsulation of charged therapeutic molecules including proteins and nucleic acids. Moreover, polymer micelles entrapping plasmid DNA and oligonucleotides have been developed as non-viral DNA delivery systems (see Kabanov, A. V., et al., Bioconjugate Chem. 6: 639-643 (1995); Kataoka, K., et al., Macromolecules 29: 8556-8557 (1996); Katayose, S., et al., Bioconjugate Chem. 8: 702-707 (1997); Kabanov, A. V., et al., Self-Assembling Complexes for Gene Delivery: From Laboratory to Clinical Trial. John Wiley and Sons, Inc., New York (1998); Nguyen, H. K., et al., Gene Ther. 7: 126-138 (2000); Lemieux, P., et al., J. Drug Target. 8: 91-105 (2000); and Kikazawa, Y. and Kataoka, K., Adv. Drug Deliv. Rev 54: 203-222 (2002)).
The references discussed above demonstrate continuing efforts to provide polymeric means of carrying or delivering chemical agents such as pharmaceuticals. Thus, there is a need in the art for improved micelle formulations for drug delivery. The present invention satisfies this need in the art.